Investigating the requirement and regulation of vascular progenitors derived from the Second Heart Field in Pharyngeal Arch Artery development

项目总结 咽弓动脉(PaaS)的不正确形成或重塑是最常见的 严重的和危及生命的先天性心脏病，如DiGeorge患者的心脏病 综合症。我们以前已经证明，PAA内皮起源于中胚层的一个亚群 先驱者被称为第二心域(SHF)。此外，血管内皮生长因子受体2 (VEGFR2)，血管发育所需的RTK在这些SHF前体细胞中高度表达为 他们贡献了PaaS。然而，在多大程度上需要SHF衍生的ECs来形成PAA，以及 调节SHF祖细胞对PaaS的贡献的机制尚不清楚。为了解决这个问题，我们 建立了一种小鼠模型，通过杂交VEGFR2GFP/+、Isl1Cre/+和 VEGFR2f/f；Rosa26mTmG/mTmG，初步假设SHF血管前体细胞VEGFR2的缺失 会导致PaaS的缺失。出乎意料的是，我们看到VEGFR2阳性的血管内皮细胞 在SHF中VEGFR2缺失的突变型胚胎。然而，咽部 与对照胚胎相比，这些突变体的内皮细胞发育不良，结构紊乱。对.的使用 CRE记者Rosa26mTmG证明，这些VEGFR2阳性的内皮细胞不是SHF起源的，而是， 来自另一个祖细胞来源，这表明内皮细胞代偿可能起作用。有趣的是， 在SHF谱系中，VEGFR2杂合子的胚胎对SHF祖细胞的贡献存在障碍。 除了在基因敲除胚胎中看到的杂乱无章的表型外，还表明可能存在 SHF中VEGFR2的单倍性不足。因此，我们假设SHF含有血管前体细胞。 对PaaS的正确形成和重塑是必不可少的。此外，我们假设内皮细胞(EC) 当SHF的贡献减弱时，来自预先存在的血管系统的祖细胞对PaaS有贡献。这里, 我们建议通过解决以下具体目标来检验这些假设：1)确定需求 和调节SHF衍生的EC在PAA发育中的作用的机制；2)确定EC的前体 当SHF派生的EC贡献受到损害时，为PAA发展做出贡献的来源(S)；3) 确定SHF来源EC贡献率为 妥协了。我们将利用条件性基因敲除小鼠模型和免疫组织化学方法来绘制命运图和 确定在PAA发育过程中对SHF血管前体细胞的需求，并确定非 胚胎PaaS中的SHF来源的内皮细胞在SHF中没有VEGFR2的表达。此外，我们将使用组合 免疫组织化学和原位杂交，以提供对调控这些血管的机制的洞察 PAA发展的先驱者。阐明PAA形成的来源和机制将导致 更好地了解冠心病的病因。

PROJECT SUMMARY The improper formation or remodeling of the Pharyngeal Arch Arteries (PAAs) are among some of the most severe and life-threatening forms of congenital heart disease, such as those seen in patients with DiGeorge Syndrome. We have previously shown that the PAA endothelium originates from a subpopulation of mesodermal progenitors termed the Second Heart Field (SHF). In addition, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an RTK which is required for vascular development is highly expressed in these SHF progenitors as they contribute the PAAs. However, to what extent SHF-derived ECs are required for PAA formation, and the mechanisms which regulate the contribution of SHF progenitors to PAAs remains unclear. To address this, we developed a mouse model to conditionally ablate VEGFR2 from the SHF by crossing VEGFR2GFP/+;Isl1Cre/+ and VEGFR2f/f;Rosa26mTmG/mTmG, with the initial hypothesis that the loss of VEGFR2 from SHF vascular progenitors would lead to the absence of PAAs. Unexpectedly, we saw VEGFR2-positive endothelium in VEGFR2GFP/f;Isl1Cre/+ mutant embryos in which VEGFR2 is deleted in the SHF. However, pharyngeal endothelium in these mutants was hypoplastic and disorganized in comparison to control embryos. The use of the Cre reporter Rosa26mTmG demonstrated that these VEGFR2-positive ECs are not of SHF origin but instead, are from another progenitor source, suggesting a possible role for endothelial compensation. Interestingly, embryos heterozygous for VEGFR2 in the SHF lineage showed an impediment in SHF progenitor contribution, in addition to the disorganized and irregular phenotype seen in the knockout embryos, suggesting a possible haploinsufficiency of VEGFR2 in the SHF. Thus, we hypothesize that the SHF contains vascular progenitors essential for proper formation and remodeling of the PAAs. Further, we hypothesize that endothelial cell (EC) progenitors from pre-existing vasculature contributes to the PAAs when SHF contribution is attenuated. Here, we propose to test these hypotheses by addressing the following specific aims: 1) To determine the requirement and mechanisms regulating SHF-derived EC contribution in PAA development; 2) To identify the EC progenitor source(s) that contribute to PAA development when SHF-derived EC contribution is compromised; 3) To determine mechanism(s) that regulate the compensation of PAA ECs when SHF-derived EC contribution is compromised. We will utilize conditional knockout mouse models and immunohistochemistry to fate map and identify the requirement of SHF vascular progenitors in PAA development, and to identify the sources of the non- SHF-derived ECs in the PAAs of embryos null for VEGFR2 in the SHF. Furthermore, we will use a combination of immunohistochemistry and in situ hybridization to provide insight on the mechanisms regulating these vascular progenitors in PAA development. Elucidating sources and mechanisms that drive PAA formation will lead to a better understanding of CHD etiology.